Ubiquitin is a protein consisting of 76 amino acids present in all eukaryotes. By the action of three kinds of enzyme groups including E1 (ubiquitin-activating enzyme)/E2 (ubiquitin-conjugating enzyme)/E3 (ubiquitin ligase), glycine residues of the C-terminal of ubiquitins form isopeptide bonds mainly with lysine residues of a substrate protein. In many cases, polyubiquitin chains consisting of ubiquitins linked with other ubiquitins are formed and function as various posttranslational modification factors (for example, see Non-Patent Document 1 and Non-Patent Document 2). Among in vivo functions of the polyubiquitin chain, for example, a selective degradation system “ubiquitin-proteasome system” established by proteasome that tracks the polyubiquitin chain as a target is most widely known. In this system, the kind of protein that will be degraded and the timing the protein is degraded are important. That is, the selectivity of a substrate is important, and the selectivity depends on a ubiquitin ligase.
Human genes are encoded with about 600 kinds of ubiquitin ligases, but the substrates of very few ubiquitin ligases have been identified. Moreover, even if it is considered that in the ubiquitin ligases whose substrate has been identified, some uncharacterized substrates are highly likely to be found. In order to understand ubiquitination involved in the control of a wide range of biological phenomenon, it is important to develop a technique that makes it possible to comprehensively search for the substrate of ubiquitin ligases with a high level of sensitivity.
So far, as an approach for identifying ubiquitinated proteins, (1) a method of comprehensive analysis in which epitope-tagged ubiquitin is overexpressed in culture cells, and mass spectrometry is performed on the proteins having undergone immunoprecipitation by tag antibodies, (2) a method in which a mutant ubiquitin ligase not having ubiquitin ligase activity is expressed, and the binding proteins are comprehensively analyzed, and the like, have been adopted. In the method (1), the number of kinds of the ubiquitinated proteins that can be identified is extremely restricted. Presumably, this is because there may be a problem with the overexpression of ubiquitins. In the method (2), a large number of binding proteins which are not substrates are also identified, and accordingly, this method is inefficient as a method for identifying a substrate. In addition, as an affinity probe for the polyubiquitin chain, Tandem ubiquitin binding entities (TUBE) obtained by the fusion of four Ubiquitin-Associated (UBA) domains have been reported (for example, see Non-Patent Document 3).
Meanwhile, in recent years, an anti-diGly antibody has been developed and greatly contributes to the identification of ubiquitinated proteins (for example, see Non-Patent Document 4). For proteomic analysis, for example, a method of performing mass spectrometry on peptides that are obtained by trypsin digestion of sample proteins is generally used. Trypsin cleaves the C-terminal of lysine and arginine, but when ubiquitinated proteins are digested with trypsin, unique peptides having a sequence (ubiquitin signature) in which two glycine residues (diGly) have formed an isopeptide bond with a lysine residue as a ubiquitination site are formed. An antibody which recognizes the ubiquitin signature is the anti-diGly antibody.